This invention pertains to the field of cancer genetics and cytogenetics. In particular, this invention pertains to the identification of nucleic acid sequences associated with a novel amplicon on human chromosome 20 which is associated with cancer. More particularly this invention pertains to the identification of a novel xe2x80x9campliconxe2x80x9d in a region of genomic nucleic acid amplification at about 20q13. These nucleic acid sequences can be used as probes in the diagnosis and prognosis of various cancers.
Chromosome abnormalities are often associated with genetic disorders, degenerative diseases, and cancer. The deletion or multiplication of copies of whole chromosomes and the deletion or amplifications of chromosomal segments or specific regions are common occurrences in cancer (Smith (1991) Breast Cancer Res. Treat. 18: Suppl. 1:5-14; van de Vijer (1991) Biochim. Biophys. Acta. 1072:33-50). In fact, amplifications and deletions of DNA sequences can be the cause of a cancer. For example, proto-oncogenes and tumor-suppressor genes, respectively, are frequently characteristic of tumorigenesis (Dutrillaux (1990) Cancer Genet. Cytogenet. 49: 203-217). Clearly, the identification and cloning of specific genomic regions associated with cancer is crucial both to the study of tumorigenesis and in developing better means of diagnosis and prognosis.
Studies using comparative genomic hybridization (CGH) have revealed approximately twenty amplified genomic regions in human breast tumors (Muleris (1994) Genes Chromosomes Cancer 10:160-170; Kalliioniemi (1994) Proc. Natl. Acad. Sci. USA 91:2156-2160; Isola (1995) Am. J. Pathol. 147:905-911). These regions are predicted to encode dominantly acting genes that may play a role in tumor progression or response to therapy. Three of these amplified regions have been associated with established oncogenes: ERBB2 at 17q12, MYC at 8q24 and CCND1 and EMS1 at 11q13. In breast cancer, ERBB2 and CCND1/EMS1 amplification and overexpression are associated with decreased life expectancy (Gaudray (1992) Mutat. Res. 276:317-328; Borg (1991) Oncogene 6:137-143). MYC amplification has been associated with lymph node involvement, advanced stage cancer and an increased rate of relapse (Borg (1992) Intern. J. Cancer 51:687-691; Berns (1995) gene 159:11-18). Clearly, the identification of additional amplified genomic regions associated with breast cancer or other tumor cells is critical to the study of tumorigenesis and in the development of cancer diagnostics.
One of the amplified regions found in the CGH studies was on chromosome 20, specifically, 20q13. Amplification of 20q13 was subsequently found to occur in a variety of tumor types and to be associated with aggressive tumor behavior. Increased 20q13 copy number was found in 40% of breast cancer cell lines and 18% of primary breast tumors (Kalliioniemi (1994) supra). Copy number gains at 20ql3 have also been reported in greater than 25% of cancers of the ovary (Iwabuchi (1995) Cancer Res. 55:6172-6180), colon (Schlegel (1995) Cancer Res. 55:6002-6005), head-and-neck (Bockmuhl (1996) Laryngor. 75:408-414), brain (Mohapatra (1995) Genes Chromosomes Cancer 13:86-93), and pancreas (Solinas-Toldo (1996) Genes Chromosomes Cancer 20:399-407). The 20q13 region was analyzed at higher resolution in breast tumors and cell lines using fluorescent in situ hybridization (FISH). A 1.5 megabase (Mb) wide amplified region within 20q13 was identified (Stokke (1995) Genomics 26:134-137); Tanner (1994) Cancer Res. 54:4257-4260). Interphase FISH revealed low-level ( greater than 1.5xc3x97) and high level ( greater than 3xc3x97) 20q13 sequence amplification in 29% and 7% of breast cancers, respectively (Tanner (1995) Clin. Cancer Res. 1:1455-1461). High level amplification was associated with an aggressive tumor phenotype (Tanner (1995) supra; Courjal (1996) Br. J. Cancer 74:1984). Another study, using FISH to analyze 14 loci along chromosome 20q in 146 uncultured breast carcinomas, identified three independently amplified regions, including RMC20C001 region at 20q13.2 (highly amplified in 9.6% of the cases), PTPN1 region 3 Mb proximal (6.2%), and AIB3 region at 20q11 (6.2%) (Tanner (1996) Cancer Res. 56:3441-3445). Clearly, definitive characterization of amplified regions within 20q13 would be an important step in the diagnosis and prognosis of these cancers.
Increased copy number of chromosome 20q in cultured cells also has been associated with phenotypes characteristic of progressing tumors, including immortalization and genomic instability. For example, increased copy number at 20q11-qter has been observed frequently in human uro-epithelial cells (HUC) (Reznikoff (1994) Genes Dev. 8:2227-2240) and keratinocytes (Solinas-Toldo (1997) Proc. Natl. Acad. Sci. USA 94:3854-3859) after transfection with human papilloma virus (HPV)16 E7 or HPV16, respectively. In addition, increased copy number at 20q13.2 has been associated with p53 independent genomic instability in some HPV16 E7 transfected HUC lines (Savelieva (1997) Oncogene 14:551-560). These studies suggest that increased expression of one or more genes on 20q and especially at 20q13.2 contribute to the evolution of breast cancer and other solid tumors. Several candidate oncogenes have been identified as amplified on 20q, including AIB1 (Anzick (1997) Science 277:965-968), BTAK (Sen (1997) Oncogene 14:2195-200), CAS (Brinkmann (1996) Genome Res. 6:187-194) and TFAP2C (Williamson (1996) Genomics 35:262-264). Clearly, definitive characterization of nucleic acid sequences in 20q13 associated with tumor phenotypes would be an important step in the diagnosis and prognosis of these cancers. The present invention fulfills these and other needs.
The present invention relates to the identification and genomic mapping of new regions of nucleic acid associated with cancer and tumorigenesis.
The invention provides a novel method for screening for the presence of an amplicon in a sample of human nucleic acid. The first step of this method provides a sample of nucleic acid derived from a human cell and a probe, where the probe comprises nucleic acid which hybridizes specifically to a nucleic acid sequence including from D20S211 through D20S120. The second step involves contacting the human nucleic acid with the probe, where the probe is contacted with the human genomic nucleic acid under conditions in which the probe binds selectively under stringent conditions to the human genomic nucleic acid to form a hybridization complex. The last step is detecting the formation of the hybridization complex. In one embodiment, the human nucleic acid can be genomic DNA, which can be isolated from a breast tumor cell. The detection step can further comprise determining the copy number of the amplicon.
In this method, the probe can also comprise a nucleic acid which hybridizes specifically to a nucleic acid sequence spanning the distance between D20S 120 and D20S211. In alternative embodiments, the probe can comprise a nucleic acid which hybridizes specifically to a STS marker selected from the group consisting of AFMa233wg1, AFM080ya1, AFM069ya1, WI-16748, WI-9939, AFMa072zb9, WI-6578, AFM224zd12, WI-9227, and AFM276xh1. The probe can also comprise a nucleic acid which hybridizes specifically to a GDB locus nucleic acid sequence selected from the group consisting of D20S211, D20S854, D20S876, D20S1044, D20S913, D20S720, and D20S 120. Alternatively, the probe can comprise a nucleic acid which hybridizes specifically to a cloned genomic nucleic acid sequence selected from the group consisting of RMC20B4097, RMC20B4103, RMC20P4016, RMC20B4130, RMC20P4185, RMC20B4188, RMC20B4109, RMC20P4010, RMC20P4028, RMC20P4003, RMC20B4099, RMC20P4018, RMC20P4069, RMC20B4121, RMC20B4087, and RMC20P4070.
In another embodiment, the probe can comprise a polymerase chain reaction primer pair capable of amplifying some or all of the nucleic acid sequence including from D20S211 through D20S120. The detection step can comprise detecting the formation of the polymerase chain reaction amplification reaction. The polymerase chain reaction primer pair can be an STS PCR primer pair selected from the group consisting of AFMa233wg1, AFM080ya1, AFM069ya1, WI-16748, WI-9939, AFMa072zb9, WI-6578, AFM224zd12, WI-9227, and AFM276xh1.
In the methods of the invention the probe can be attached to a solid surface and the attached probe can be a member of a nucleic acid array. The human nucleic acid can be labeled with a detectable composition. The detectable composition can be fluorescein or Texas red. In an alternative embodiment, the probe is labeled with a detectable composition. The method can further provide nucleic acids from a reference cell, wherein the reference cell nucleic acid is contacted with the probe before or simultaneously with the human genomic nucleic acid. The method can further provide Cot-1 DNA, wherein the Cot-1 DNA is hybridized to the human genomic nucleic acid before contacting the human genomic nucleic acid with the probe.
The invention also provides a nucleic acid probe for screening for the presence of an amplicon in a sample of human genomic nucleic acid, comprising a nucleic acid which hybridizes specifically to a nucleic acid sequence including from D20S211 through D20S120. Alternatively the probe can comprise nucleic acid which hybridizes specifically to: the nucleic acid sequence spanning the distance between D20S120 and D20S211; or, a STS marker selected from the group consisting of AFMa233wg1, AFM080ya1, AFM069ya1, WI-16748, WI-9939, AFMa072zb9, WI-6578, AFM224zd12, WI-9227, and AFM276xh1; or, a GDB locus nucleic acid sequence selected from the group consisting of D20S211, D20S854, D20S876, D20S1044, D20S913, D20S720, and D20S120; or, a cloned genomic nucleic acid sequence selected from the group consisting of RMC20B4097, RMC20B4103, RMC20P4016, RMC20B4130, RMC20P4185, RMC20B4188, RMC20B4109, RMC20P4010, RMC20P4028, RMC20P4003, RMC20B4099, RMC20P4018, RMC20P4069, RMC20B4121, RMC20B4087, and RMC20P4070.
In another embodiment, the probe can comprise a polymerase chain reaction primer pair capable of amplifying some or all of the nucleic acid sequence including from D20S211 through D20S120. The polymerase chain reaction primer pair can be an STS PCR primer pair selected from the group consisting of AFMa233wg1, AFM080ya1, AFM069ya1, WI-16748, WI-9939, AFMa072zb9, WI-6578, AFM224zd12, WI-9227, and AFM276xh11.
The invention also provides a kit for screening for the presence of an amplicon in a sample of human nucleic acid, the kit comprising a compartment which contains a probe, wherein the probe comprises nucleic acid which hybridizes specifically to a nucleic acid sequence including from D20S211 through D20S120. Alternatively, the probe can comprise nucleic acid which hybridizes specifically to: sequences which span the distance between D20S120 and D20S211; or, a STS marker selected from the group consisting of AFMa233wg1, AFM080ya1, AFM069ya1, WI-16748, WI-9939, AFMa072zb9, WI-6578, AFM224zd12, WI-9227, and AFM276xh1; or, a GDB locus nucleic acid sequence selected from the group consisting of D20S211, D20S854, D20S876, D20S1044, D20S913, D20S720, and D20S120; or, a cloned genomic nucleic acid sequence selected from the group consisting of RMC20B4097, RMC20B4103, RMC20P4016, RMC20B4130, RMC20P4185, RMC20B4188, RMC20B4109, RMC20P4010, RMC20P4028, RMC20P4003, RMC20B4099, RMC20P4018, RMC20P4069, RMC20B4121, RMC20B4087, and RMC20P4070.
In another embodiment, the probe of the kit can comprise a polymerase chain reaction primer pair capable of amplifying some or all of the nucleic acid sequence including from D20S211 through D20S120. The polymerase chain reaction primer pair can be an STS PCR primer pair selected from the group consisting of AFMa233wg1, AFM080ya1, AFM069ya1, WI-16748, WI-9939, AFMa072zb9, WI-6578, AFM224zd12, WI-9227, and AFM276xh1. The probe can be a cloned human nucleic acid, and the cloned human genomic nucleic acid can be attached to a solid surface. The attached probe can be a member of a nucleic acid array. The kit can further comprises instructional material that indicates that the detection of greater than two amplicon copies in a cell can be diagnostic or prognostic of cancer or tumorigenesis.
A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification, the figures and claims.
All publications, GenBank Accession references (sequences), patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.